Biological fluid assay methods

ABSTRACT

A method to assess the level of folate in a biological sample comprises: 
     providing said sample with glycine N-methyltransferase (GMT) and with an excess of S-adenosyl methionine (SAM) and of glycine; 
     providing a control which contains no folate with said GMT and excess SAM and glycine in comparable amounts to those provided to the sample; and 
     comparing the concentration of at least one product formed in the sample with the concentrations of said product formed in the control, 
     whereby the difference in levels of said product in the sample as compared to the control is directly proportional to the level of folate in the sample.

This application claims priority under 35 United States Code §119(e)from provisional application Ser. No. 60/129,730 filed Apr. 16, 1999,the contents of which are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to diagnostic methods for conditions which arecharacterized by abnormal levels of certain metabolites, in particular,folates, and to simplified methods of detection. The improved methods ofthe invention provide rapid and accurate assessment of theconcentrations of folate and other analytes.

1. Background Art

Folates are critical co-factors in methyl transfer reactions.Abnormalities of folate levels in biological fluids such as blood andplasma are indicative of conditions that are characterized byinappropriate or inadequate transfer of methyl groups. Thus, it isimportant to be able to provide an accurate measure of folate levels insuch fluids and to compare them to those expected in normal individuals.Low folate concentrations are associated with megaloblastic anemia andreduced DNA synthesis, as well as cardiovascular disease. Birth defectsmay also result.

Folate can be measured efficiently using the methods of the invention.The invention also provides an improved method to detect H₂O₂ in thepresence of peroxidase

2. Disclosure of the Invention

The invention method for determining levels of folate in biologicalfluids takes advantage of the ability of folate to inhibit the methyltransfer reaction whereby glycine is converted to sarcosine. The enzymewhich carries out this conversion, glycine N-methyltransferase (GMT) canbe isolated from liver or pancreas. In the assay systems of theinvention, glycine and S-adenosyl methionine (SAM) are the reactants andsarcosine and S-adenosyl homocysteine (SAH) are the products. Theeffectiveness of GMT in converting these reactants is dependent onfolate concentration in vivo.

The folate in the sample can be converted to a form (to the extent itdoes already exist in said form) whereby it inhibits the ability of GMTto form the foregoing products. The folate in the sample is firstconverted to folypoly glutamate (FPG) which is an inhibitor of the GMT.Yeo, E-J, et al., J. Biol. Chem. (1999) 274:37556-37564. Under theseconditions, the greater the amount of folate in the sample, the lessproducts formed.

In the invention method, the levels of product are typically measured.As the levels of reactants are provided in excess, although it istheoretically possible to measure the diminution in reactants, this isless desirable as it is, of course, more difficult to detect smalldifferences in large concentrations than to measure a difference fromzero. However, it is not impossible, and such measurements are withinthe scope of the invention as well.

Either the levels of SAH or of sarcosine can be measured or both. Avariety of methods is available in each case. Preferred embodiments forthe measurement of SAH, however, include its conversion to homocysteineand subsequent lysis of homocysteine to obtain readily detectableproducts as further described below. In the case of sarcosine, aconvenient method is the use of a specific oxidase which, in thepresence of this amino acid, generates hydrogen peroxide which can alsobe readily detected by a variety of methods known in the art, preferredembodiments of which are illustrated below. Thus, in one aspect, theinvention is directed to:

A method to assess the level of folate in a biological fluid samplewhich method comprises

providing said sample with glycine N-methyltransferase (GMT) and with anexcess of S-adenosyl methionine (SAM) and of glycine;

providing a control which contains no folate with said GMT and excessSAM and glycine in comparable amounts to those provided to the sample;and

comparing the concentration of at least one product formed in the samplewith the concentrations of said product formed in the control,

whereby the difference in levels of said product in the sample ascompared to the control is directly proportional to the level of folatein the sample.

In the foregoing method, either sarcosine or SAH levels may be measured.The concentrations of products will be diminished in the sample ascompared to the control. Pretreatment of the sample with an enzyme whichconverts folate to an inhibitor of GMT, FPG, set forth above, isdesirable. This enzyme, folypoly glutamate synthetase (FPGS) is readilyavailable in the art.

In another aspect, the invention is directed to a particularlyconvenient method for the endpoint of sarcosine measurement. It has beenfound that a particularly convenient method to measure hydrogen peroxidein analytical samples, including biological fluids, is to treat thesample containing the peroxide with both peroxidase and a dialkylphenylene diamine. The resultant is a colored compound which can bemeasured spectrophotomctrically. As will be evident, since a number ofprotocols are available which result in the generation of hydrogenperoxide, for example the treatment of substrates with their pertinentcorresponding and specific oxidases, the combination of peroxidase anddialkyl phenylene diamines has a broad range of utility.

MODES OF CARRYING OUT THE INVENTION

The invention is directed, in one embodiment, to the measurement offolates in biological fluids. Suitable biological fluids are mostcommonly blood, serum and plasma, although other fluids may be ofinterest as well, such as cerebral spinal fluid, urine, and other bloodfractions. Measurement in plasma or serum is preferred.

By “folates” is meant folic acid and its salts as well as the dihydroand teterahydro form. In order to ensure uniformity of results, it maybe desirable to add a reducing agent to the reaction mixtures in orderto ensure that all of the folates are in the same oxidation state—i.e.,the teterahydro folate (THF) form. However, it is believed that thepredominant form, almost to the exclusion of the others, is biologicalsystems is tetrahydrofolate, and in particular,5-methyltetrahydrofolate. It is this form, specifically, in combinationwith glutamate which acts as an inhibitor of GMT endogenously.

By way of background, it is believed that the function of GMT in theliver and pancreas is to diminish undesirably high concentrations of SAMby creating a “sink” in the form of sarcosine, which has no knownfunction. However, this enzyme is regulated by folate; when high levelsof methylated THF are present, combination with glutamate occurs and theresulting compound is a powerful GMT inhibitor. The present inventiontakes advantage of the ability of folates to inhibit GMT using thismechanism.

For use in the present invention, GMT can be isolated from pancreas orliver or theoretically could be produced recombinantly. If the enzyme isisolated from native sources, it should first be treated to disassociateit from any endogenous folate. This can be effected by treating theenzyme with an anion exchange column and effecting a separation using,for example, a gel exclusion method.

In the methods of the invention, glycine N-methyltransferase (GMT)catalyzes the reaction:

glycine+SAM→sarcosine+SAH.

The GMT is purified from any convenient source. A method forpurification of to the enzyme from rat liver is described in theexamples below; also described is a method to obtain the enzyme free ofinhibitor by treating the isolated GMT on an anion exchange column andseparating the dissociated folate by gel filtration.

To carry out the method, both control and sample are incubated withfolate-free GMT after treatment with glutamic acid in the presence ofthe enzyme FPGS which converts any folate into an inhibitor of GMT, FPG.Either sarcosine or SAH can be measured, the amounts or concentrationsof product present in the sample will be less than those of the controlin a degree proportional to the concentration of folate in the sample.

Thus, the sample and the control are treated with glutamine and thesynthetase FPGS to convert the folate to an inhibitor (FPG) of the GMT.In this embodiment, the sample is incubated with the glutamine and FPGSin suitable amounts prior to or concomitant with, treatment with GMT.The remainder of the assay is conducted according to the alternativesoutlined below. Measurement of SAH or sarcosine provides a measure ofthe GMT activity which is, in turn, inversely proportional to the levelof inhibitor, and thus inversely proportional to the level of folate inthe sample.

Detection of SAH

In a preferred method for detection of SAH, the reaction mixtures aretreated with S-adenosyl homocysteinase (SAHase) which converts SAH tohomocysteine. Homocysteine can then be measured by any method convenientin the art. However, a particularly preferred embodiment employs arecombinant homocysteinase (HCYase), preferably HYase™, which isespecially specific for homocysteine. This enzyme is described in detailin PCT publication No. WO99/05311 published Feb. 4, 1999, andincorporated by reference. The use of this particular HYase™homocysteinase has the advantage of substantially and essentiallyeliminating any background products which might be formed frommethionine or cysteine present in the sample. The products ofhomocysteine catalysis by this enzyme are ammonia, hydrogen sulfide, andα ketobutyrate. Any of these products may be measured, but it is mostconvenient to measure the hydrogen sulfide by the use of an oxidizingagent in the presence of any dialkyl phenylene diamine, mostconveniently the di-N-butyl embodiment, DBPDA. The complex that isformed between hydrogen sulfide and the dialkyl phenylene diamine in thepresence of an oxidizing agent is both chromogenic and fluorescent, andeither property can be used for detection. Use of the fluorescenceproperty appears slightly more sensitive.

Thus, in one preferred embodiment, the reaction mixture is treated withSAHase and recombinant homocysteinase HYase and with the chromogen DBPDAand an oxidizing agent such as ferric ion. Either the absorbance atabout 675 nm or the fluorescence upon excitation at about 665 nm andemission at 690 nm is measured.

Measurement of Sarcosine

The levels of sarcosine product can conveniently be measured by use ofsarcosine-specific enzymes. A particularly preferred sarcosine-specificenzyme is sarcosine oxidase Santos, F., et al., Electrophoresis (1995)16:1898-1899, which generates hydrogen peroxide. Dialkyl phenylenediamines are also chromogenic in the presence of hydrogen peroxide uponbreakdown with peroxidase; alternatively, a colored product can beobtained using other reagents, such as 4-chloro-1-naphthol andorthotoluidine.

Measurement of Peroxide

The are many biological reactions, especially oxidase reactions, whichare specific for individual substrates and which result in theproduction of hydrogen peroxide. Another aspect of the invention is animproved method to measure the concentration of hydrogen peroxide whichmethod comprises adding to the sample where in the peroxide is to bemeasured both peroxidase, such as horseradish peroxidase, and a dialkylphenylene diamine. A preferred compound is DBPDA. Illustrative amountsof these reagents are described in the examples below.

The product of the reaction can be measured spectrophotometrically.Alternatively, a fluorescent product is formed which can be detectedaccordingly.

The following examples illustrate but do not limit the invention.

Preparation A Purification of Rat Liver Glycine N-Methyltransferase(GMT)

The method is a modified form of that described by Ogawa, H., J. Biol.Chem. (1982) 257: 3447-3452; Yeo, E. J., J. Biol. Chem. (1992) 267:24669-24674.

Rats were killed by decapitation and the livers quickly removed andchilled on ice. About 400 g liver was homogenized in a blender with1.21. of 10 mM buffer, pH 7.2, containing 1 mM EDTA. After adjusting topH 5.5 by dropwise addition of 1.0 M sodium acetate, the precipitate wasremoved by centrifugation at 10,000×g for 15 min. The clear supernatantsolution was recovered and adjusted to pH 7 with 2 N KOH.

The supernatant was made 1% ammonium sulfate and the mixture was allowedto stand for 30 min. with gentle stirring. After centrifugation at10,000 g for 30 min, the supernatant was recovered and treated with 8 gof ammonium sulfate per 100 ml of supernatant. After 30 min, the mixturewas centrifuged as above, and the pellet was recovered, dissolved anddialyzed overnight.

The dialyzed enzyme preparation was divided into equal parts and eachwas put onto a column of DEAE-Sepharose (5×25 cm) equilibrated with 10mM buffer, pH 7.2, containing 1 mM EDTA. The pass-through fractions werecombined and ammonium sulfate added at 30 g/100 ml. After standingovernight, the pellet was collected by centrifugation and dissolved inbuffer.

The enzyme solution containing 1.0 M ammonium sulfate was applied to aPhenyl Sepharose 6 FF (5×25 cm) column prewashed with 10 mM buffer, pH7.2, containing 1.0 M ammonium sulfate. The bound GMT protein was elutedin a linear gradient of decreasing the ammonium sulfate concentration,using 10 mM buffer, pH 7.2. The peak showing the activity was collectedand dialyzed overnight.

Preparation B Preparation of Apo GMT

Folate was removed from the GMT prepared as in Preparation A. The enzymesolution prepared in Preparation A was passed over and anion exchangecolumn to dissociate the folate ligand, followed by centrifugal gelexclusion using standard methods.

Preparation C Preparation of the Inhibitor FPG

FPG was prepared from serum or from standard 5-methyltetrahydrofolatesolutions by treating with recombinant human FPGS (Paresh, C., ProteinExp & Pur (2000) 18: 36-45. Serum or 5-methylTHF was incubated with 3 mgpurified recombinant human FPGS (0.1 μM) in the presence of 15 mMglutamic acid, 10 mM MgCl₂, 5 mM ATP, 20 mM KCl, 100 mMβ-mercaptoethanol, 250 μg BSA, 100 mM Tris-HCl, pH 8.7, in a totalvolume of 500 μl at 37° C. for 1.0 hour.

EXAMPLE 1 Determination of Folate by Inhibition of GMT by FPG

GMT (0.95 μg) as prepared in Preparation A was treated to associate withFPG in a reaction mixture of standard (1-100 nM FPG) or test serumsample containing 20 mM Tris-HCl, pH 9.0 and 5 mM DTT. Various dilutionsof Preparation C (FPG prepared from standard methylTHF or from serum)were included in an 800 μl reaction mixture which was incubated for 15min. at 25° C.

After the GMT and FPG (either standards or derived from serum) wereallowed to associate, the volume was brought in each sample to 1000 μlwith 100 μl 5 mM glycine and 100 μl 1 mM SAM. This reaction system alsoincluded the assay mixture of 10 μl recombinant SAHase (2.0 units/ml)and recombinant HYase (300 units/ml). It was incubated at 37° C. for 30min. During this period, any SAH product is converted to homocysteineand then to hydrogen sulfide, ammonia and pyruvate.

After the 30 min. incubation, 50 μl 40 mM DBPA and 40 mM K₃Fe (CN)₆ wereadded and the reaction mixture was incubated at 37° C. for 10 min. andread at 675 nm.

EXAMPLE 2 Alternate Assessment Method

A. Assessment of folate in serum was carried out as set forth in Example1 except that instead of including SAHase and HCYase in the reactionmixture, the reaction system contains 10 μl sarcosine oxidase (SOX) (4mg/ml) peroxidase (2 mg/ml) and 10 μl of 4-chloro-1-naphthol (2% W/V indiethylene glycol) and 20 μl of o-toluidine (saturated solution in 7%acetic acid). The incubation was again at 37° C. and the optical densitywas read.

B. The assay of paragraph A was carried out except that no4-chloro-1-naphthol or o-toluidine were added to the reaction mixture.Instead, 5 mM DBPDA was included, and the optical density read at 558 nmat the end of the 30 min. incubation.

What is claimed is:
 1. A method to assess the level of folate in abiological fluid sample which method comprises providing said samplewith glycine N-methyltransferase (GMT) and with an excess of S-adenosylmethionine (SAM) and of glycine; providing a control which contains nofolate with said GMT and excess SAM and glycine in corresponding amountsto those provided to the sample; and comparing the concentration of atleast one product formed in the sample with the concentrations of saidproduct formed in the control, whereby the difference in levels of saidproduct in the sample as compared to the control is directlyproportional to the level of folate in the sample.
 2. The method ofclaim 1 wherein the product detected is S-adenosyl homocysteine (SAH).3. The method of claim 2 wherein said SAH is measured by a method whichcomprises treating the sample with SAHase to convert SAH to homocysteineand with homocysteinase (HCYase) and measuring the concentration of atleast one product obtained by the reaction of HCYase with saidhomocysteine.
 4. The method of claim 3 wherein the product measured isH₂S.
 5. The method of claim 4 wherein said measuring comprises treatingthe sample with a dialkyl phenylenediamine and an oxidizing agent. 6.The method of claim 5 wherein the dialkyl phenylene diamine is DBPDA andthe oxidizing agent is ferric ion.
 7. The method of claim 2 wherein thesample and control are incubated with said GMT in the presence offolypoly glutamate synthetase (FPGS) and glutamic acid in concentrationseffective to convert any folate in the sample to folypoly glutamate(FPG) wherein the concentration of SAH in the sample is less than theSAH present in the control to a degree directly proportional to theconcentration of folate in the sample.
 8. The method of claim 1 whereinthe product measured is sarcosine.
 9. The method of claim 8 wherein saidsarcosine is measured by treating said sample with sarcosine oxidase andmeasuring the level of at least one product of the reaction of sarcosinewith sarcosine oxidase.
 10. The method of claim 9 wherein the productmeasured is hydrogen peroxide.
 11. The method of claim 10 wherein saidmeasuring comprises treating with peroxidase.
 12. The method of claim 11wherein said measuring further comprises providing the sample with adialkyl phenylene diamine.
 13. The method of claim 12 wherein thedialkyl phenylene diamine is DBPDA.
 14. The method of claim 10 whereinsaid measuring comprises providing peroxidase and 4-chloro-1-naphtholand o-toluidine.